Pluripotent stem cells (PSCs) have the potential to produce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evidences suggest that telomere length represents another important internal factor in maintaining stem cell pluripotency. Telomere length homeostasis and its structural integrity help to protect chromosome ends from recombination, end fusion, and DNA damage responses, ensuring the divisional ability of mammalian cells. PSCs generally exhibit high telomerase activity to maintain their extremely long and stable telomeres, and emerging data indicate the alternative lengthening of telomeres (ALT) pathway may play an important role in telomere functions too. Such characteristics are likely key to their abilities to differentiate into diverse cell types in vivo. In this review, we will focus on the function and regulation of telomeres in ESCs and iPSCs, thereby shedding light on the importance of telomere length to pluripotency and the mechanisms that regulate telomeres in PSCs.
Animals ; Humans ; Models, Biological ; Pluripotent Stem Cells ; metabolism ; Telomerase ; metabolism ; Telomere ; metabolism ; Telomere Homeostasis

OBJECTIVE: The patient monitors were used to explore the alarm fatigue in a cardiac care unit and to investigate the awareness and reaction of nurse to alarms. METHODS: A semi-structured survey was taken to acquire nurses' feeling and knowledge about monitoring alarm. Three full-time researchers were scheduled to track the alarms with annotations, and analyze the alarm data of 12 patient monitors using central monitoring system. RESULTS: A total of 72 310 unique alarms occurred in the 67-day study period. About 75.7% of them were physiological alarms and less than 10% of medium-low alarms were false positives. The average alarm rate was 128 alarms/patient-day. CONCLUSIONS There remains alarm fatigue in CCU, the alarm accuracy has improved than the past by applying new technologies. In some cases, clinicians will pay more attention to trend alarm and combination alarm.
Arrhythmias, Cardiac ; Clinical Alarms ; Electrocardiography ; Humans ; Monitoring, Physiologic ; Surveys and Questionnaires

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different organisms. Current CRISPR-activation (CRISPRa) platforms often require multiple components because of inefficient transcriptional activation. Here, we fused different phase-separation proteins to dCas9-VPR (dCas9-VP64-P65-RTA) and observed robust increases in transcriptional activation efficiency. Notably, human NUP98 (nucleoporin 98) and FUS (fused in sarcoma) IDR domains were best at enhancing dCas9-VPR activity, with dCas9-VPR-FUS IDR (VPRF) outperforming the other CRISPRa systems tested in this study in both activation efficiency and system simplicity. dCas9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of dCas9-VPR. These findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.
Humans ; Transcriptional Activation ; RNA, Guide, CRISPR-Cas Systems ; Gene Expression Regulation ; CRISPR-Cas Systems/genetics*

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
APOBEC-1 Deaminase ; genetics ; metabolism ; Base Sequence ; Blastomeres ; cytology ; metabolism ; CRISPR-Cas Systems ; Embryo, Mammalian ; metabolism ; pathology ; Female ; Fibroblasts ; metabolism ; pathology ; Gene Editing ; methods ; Gene Expression ; HEK293 Cells ; Heterozygote ; Homozygote ; Humans ; Point Mutation ; Primary Cell Culture ; Promoter Regions, Genetic ; Sequence Analysis, DNA ; beta-Globins ; genetics ; metabolism ; beta-Thalassemia ; genetics ; metabolism ; pathology ; therapy

Adenine ; Animals ; Gene Editing ; Mice ; Zygote ; metabolism

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation